Mechanism of Voltage Sensor Probe Technology and VSP Applications

Voltage Sensor Probes (VSPs) are a Fluorescence Resonance Energy Transfer (FRET)-based voltage-sensing assay technology, which measures changes in cellular membrane electrical potential. (reference figure here). The probes enable detection and measurement of rapid (sub-second), dynamic changes in membrane voltage and quickly report them as optical (fluorescence) signals from living cells (1-5). As a result, VSPs are ideal for high-throughput screening (HTS) of compounds that modulate ion channels or change the membrane potential. VSPs have already successfully screened more than 32,000 test compounds a day (1).

VSPs use a FRET pair that is composed of one of two highly fluorescent, mobile, voltage-sensitive acceptor oxonols [DiSBAC2(3) or DiSBAC4(3)] and a fluorescent, membrane-bound coumarin-phospholipid FRET donor (CC2-DMPE). When the two dyes are loaded into the cellular plasma membrane, they associate with different parts of the membrane. The CC2-DMPE donor binds specifically to only one face of the plasma membrane, so it is relatively stationary. CC2-DMPE has a 400-nm excitation wavelength and a 460-nm emission wavelength. Each of the mobile oxonol acceptors is a negatively charged hydrophobic ion that has two binding sites, one on each side of the plasma membrane. As a result, although each oxonol initially binds to one face of the plasma membrane, each will rapidly diffuse to the other side (on a subsecond time scale) in response to changes in the electrical potential in the cell membrane, establishing a new equilibrium corresponding to the new membrane potential. Thus, each oxonol "senses" and responds to voltage changes. Each oxonol acceptor has an excitation wavelength of 540 nm and an emission wavelength of 580 nm.

Figure 1. The VSP FRET pair consists of a mobile, voltage-sensitive acceptor and an outer membrane-bound donor. In resting cells (with a relatively negative internal potential), both members of the FRET pair bind to the outer surface of the cell membrane, resulting in efficient FRET. When the cells are depolarized, however, the donor remains on the outer surface, but the mobile acceptor rapidly translocates to the inner surface of the cell membrane, resulting in diminished FRET. The normalized assay ratio (the ratio of the donor emission to acceptor emission) reports changes in potential and is high in polarized cells and low in depolarized cells.

VSP Applications

VSPs can report on any target that changes the membrane potential of the cell, including ligand-gated ion channels, Na, K, Ca, Cl channels and transporters (1–6). VSPs allow development of fluorescence-based high-throughput assays that measure the affinity of reference compounds with excellent correlation to patch-clamp data for voltage-gated NaV channels (7–9) and GABAA ligand-gated channels (10). Representative data seen in Figure 2 demonstrate VSP sensitivity for reporting on voltage-gated sodium channel-mediated depolarization, and on the effects that two compounds (a sodium channel opener and a sodium channel blocker) have on sodium channels.

Figure 2. Identification of NaV agonist and antagonist with a VSP assay.

A. Signal intensity changes for each dye in the FRET pair, and emission ratio changes following sodium addition to cells previously maintained in a sodium-free extracellular buffer. Sodium influx into the cells leads to a dramatic decrease in membrane potential, as reflected in the rapid decrease in oxonol emission. This decrease is accompanied by an increase in coumarin emission, causing a sharp increase in the emission ratio.

B. Depicts two dose-response curves: (1) an ascending curve demonstrating that the cells’ membrane potential significantly decreases when resting cells are exposed to increasing concentrations of a sodium channel opener and (2) a descending curve indicating that the cells’ membrane potential rapidly returns to the resting state when stimulated cells are exposed to increasing concentrations of a sodium channel blocker. This demonstrates the utility of the VSP assay for detecting both openers and blockers of this voltage-gated sodium channel. Using this cell line, a high-throughput screen of 700,000 molecules has been run in a 384-well assay format using the VIPR® II reader to identify novel sodium channel blockers.